Low elasticity films for microfluidic use

ABSTRACT

Microfluidic circuit elements, such as a microvalve, micropump or microvent, formed of a microcavity divided by a diaphragm web into a first subcavity bounded by a first internal wall and a second subcavity bounded by a second internal wall, where the diaphragm web is characterized as a thin film having a first state contacting the first internal wall and a second state contacting the second internal wall and exhibiting essentially no elasticity in moving between the first state and the second state, the thin film web having been stretched beyond its yield point before or during use are provided. The disclosed elements enable faster and more efficient cycling of the diaphragm in the microcavity and increases the diaphragm surface area. In a preferred embodiment, the microfluidic circuit element is pneumatically driven and controls the motion of fluids in a microassay device.

BACKGROUND

1. Technical Field

This disclosure is generally directed to diaphragm technologies formicrovalves, micropumps and other pneumatic fluidic elements for use inmicroassay devices, and to their methods of manufacture.

2. Description of the Related Art

Microassay cartridges have found increasing use as devices fordiagnostic assays. The devices described by Wilding in U.S. Pat. No.5,304,487 consisted of “mesoscale” channels and chambers formed onreusable silicon substrates that were infused with fluid reagents fromoff-cartridge syringe pumps. Little consideration was given to on-boardfluid handling and control. However, practical commercial applicationshave led in the direction of “consumable” cartridges—disposable, singleuse “sample-to-answer” cartridges that are self-contained for allreagents needed for a particular assay or panel of assays.

Microscale means for handling fluids include mechanical hydraulicsystems such as piston driven devices, electrical hydraulic systems suchas electrokinetic pump and valve devices, and pneumohydraulic systems.Of these, those systems with pneumatic actuators and control surfaceshave proven to be particularly practical in controlling microscale fluidflows.

One well known class of fluidic devices having a pneumatic interface ismanufactured by the Assignee, Micronics, Inc. (Redmond, Wash.). Controlof fluid flow in microfluidic channels is achieved with a MICROFLOW®system pneumatic controller that operates miniature valves in a plasticcartridge according to programmable valve logic. Diaphragms separate thepneumatic side and the hydraulic side of the cartridges; i.e., the valvediaphragms are interface elements for converting pneumatic controlpulses into starting and stopping fluid flow. Cartridges are formed bybuilding up laminations, layer by layer, with channels and chamberssealed between capping overlayers. In this way, complex fluidic circuitsare formed.

To form a fluidic circuit by conventional fabrication, a layer of anelastic material is sandwiched as a laminate between body layers, andpneumatic and hydraulic channels and chambers are formed in the apposinglayers on either side of the elastic layer, such that the pneumaticworkings and the hydraulic workings of the cartridge are separated by adiaphragm layer. Diaphragms formed of polyurethane, polyimide, and PDMShave been favorites for this method.

Miniature pump elements, for example, are needed to achieve the fullestbenefit of fluidic microcircuitry technologies, which find numerousapplications such as in diagnostics and in life sciences more generally.Diaphragm-driven pumps are advantageous because of the absence ofmechanical seals and lubricant, and their sanitary features.

Although miniature pumps were generically hinted at by Wilding (forexample in U.S. Pat. Nos. 5,304,487 and 5,498,392), the disclosuresthemselves were not sufficient to enable fluidic microcircuitry pumpsand valves. Cited by Wilding was Van Lintel [1988, “A PiezoelectricMicropump Based on Micromachining of Silicon,” Sensors and Actuators,15:153-167], which relates to silicon-based microelectromechanical(MEMS) structures. However, silicon is known to have a very high Young'smodulus (about 100 GPa); therefore a silicon diaphragm pump willgenerally have a very low compression ratio ε defined by:

ε=(ΔV+V ₀)/V ₀

where ΔV is the stroke volume and V₀ is the deadspace volume, i.e., thevolume of fluid that is not displaced from the pumping chamber during anejection stroke. Thus disadvantageously, these devices may not beself-priming in operation when used with liquids.

Representative art related to siliceous diaphragm pumps includes U.S.Pat. Nos. 5,759,014, 6,390,791 and 7,749,444. Similar issues are seenwith the rigid polymeric diaphragm members of U.S. Pat. No. 7,832,429and more generally where the diaphragm member resists deformation due tomechanical stiffness.

There has been greater interest in elastomeric diaphragm materialsbecause of the higher compression ratio, which offers the advantage ofself-priming in fluidic operations, and larger displacement volume. Forexample, polydimethylsiloxane (PDMS) and silicones may be used asdiaphragm materials. Latex rubber and amorphous polyurethanes have alsobeen used. Elastomeric materials that obey Hooke's law have theadvantage that the diaphragm returns to its original shape in therelaxed state, but this is advantageous only for some applications, andcan be associated with reduced chemical resistance and increasedpermeability.

Representative art related to microvalves includes U.S. Pat. No.4,304,257 (the '257 valve), in which a soft, resilient, polyurethanesheet is clamped over flow channels formed in a hard acrylic body. Afluid path between two discontinuous fluid channels is opened and closedby actuating pistons which mechanically flex a part of the sheet. Atenting action on the sheet is associated with valve opening; valveclosing is associated with spring return of the resilient sheet to aclosed position. The sheet is flexed mechanically between the twopositions by a solenoid-operated rod having an embedded attachment tothe sheet over the valve seat, such that the sheet contacts the seatwhen closed and the sheet is pulled into an aperture overlying the valveseat to open the valve.

According to the teachings of U.S. Pat. No. 4,848,722, the '257 valvehas several disadvantages. In addition to delicacy of mechanicalsolenoid operation and need for fine adjustment, the membrane issubjected to great stresses with the risk of permanent stretch (i.e.,permanent deformation or pinching past its yield point). By virtue ofthe concave contact surface for the membrane, the sealing area ismaximized, but disadvantageously, a non-zero and significant volume ofthe valve cavity must be filled before fluid begins to flow.

In expired U.S. Pat. No. 4,848,722 (the '722 valve), a pressure orvacuum source is used to urge a flexible sheet such as biaxiallyoriented polyethylene terephthalate (BoPET) into a stop-flow position inwhich apertures formed by the channels (3,4) in the valve seat areclosed and an open position in which the apertures are fluidlyconfluent. The step land (FIG. 9: 62) of the valve seat is contacted bysheet (8) when the valve is closed. The sheet is glued to the pneumaticside of the valve.

U.S. Pat. No. 4,869,282 describes a micromachined valve having adiaphragm layer sandwiched between two rigid layers forming the valvecavity. The diaphragm layer is formed of polyimide and is deflected byan applied pneumatic pressure in a control circuit to close the valve.Diaphragm motion is limited to avoid overstressing the polyimide layer.

Expired U.S. Pat. No. 5,660,370 (the '370 valve) describes a valve (FIG.1: 1) having flexible diaphragm (2) and flat valve seat formed of arigid layer in which two holes are formed, each hole defining an openingto a fluidic channel (3,4) in an underlying layer, where the holes areseparated by a valve sill. The diaphragm is made of polyurethane orsilicone. The valve (5) is opened by pneumatically exercising thediaphragm. To avoid the tendency of the sheet to become stressed beyondits yield point, a flat valve seat is used to minimize the requiredrange of diaphragm motion. This also reduces the deadspace volume of thevalve.

A similar structure is seen in U.S. Pat. No. 5,932,799 to YSI Inc.,which teaches a fluidic microcircuitry analyzer having a plurality ofpolyimide layers, preferably KAPTON® film, directly bonded togetherwithout adhesives and a flexible pneumatically actuated diaphragm memberfor controlling fluid flow.

WO Publ. No. 2002/081934 to Micronics, Inc., published Oct. 17, 2002,describes a laminated valve having an elastomeric diaphragm. Thesevalves, which were termed “peanut valves”, admit fluid across the valvesill under negative pressure, and are closed when positivelypressurized. Advantageously, the valve cavity is formed with a contouredwaist to minimize deadspace volume.

U.S. Pat. No. 7,445,926 to Mathies describes a laminate with a flexiblediaphragm layer sandwiched between hard substrates. Pneumatic channelsand fluid channels are formed on opposite sides of the diaphragm layer(cf., FIG. 1 of the reference), so that the diaphragm is the activevalve member. The diaphragm material disclosed is a 254 micrometer PDMSmembrane. The valve body is typically a solid such as glass.

US Pat. Appl. Nos. 2006/0275852 and 2011/0207621 to Montagu describe afluidic cartridge for biological assays. The cartridge includes a moldedbody defining flow passages. A latex diaphragm and a canned diaphragmpump are shown (cf., FIG. 5 of the reference). The “rolling elasticdiaphragm pump” member (3) is inserted into the cartridge as apre-formed subassembly and is commercially available (Thomas Pumps,Model 1101 miniature compressor, Sheboygan, Wis. 53081). Valves aremechanically actuated using a stepper motor. Thus the valves have thedisadvantage of requiring sensitive and meticulous adjustment for properoperation. Other elastomeric valve and pump constructs are known.Examples of silicone valve construction include U.S. Pat. Nos.5,443,890, 6,793,753, 6,951,632 and 8,104,514, all of which illustratesoft lithographic processes (cf., U.S. Pat. Nos. 7,695,683 and8,104,497) for forming valves and pumps. PDMS may be used to formdiaphragms and pump bodies. Latex rubber and amorphous polyurethaneshave also been used as diaphragm materials, but chemical resistance maynot be sufficient for some applications.

Diaphragm members having toughness, solvent resistance and capable ofbeing shaped into yield-in-place diaphragms have not previously beendemonstrated. Advantageously, a solvent-resistant diaphragm that yieldsto form a pre-shaped diaphragm member has application in pumps andvalves used for pumping suspensions of particulates, and for replacingelastomeric diaphragms such as polyurethane which may leak when exposedto caustics, chaeotropes, or solvents, thus permitting use of solventssuch as ethanol, formamide and dimethylsulfoxide, e.g., for lowering theoperating temperature requirements during PCR. Yield-in-place diaphragmshave increased pump stroke ejection volumes, leading to faster circuitresponse, and improved flow of particulate solutions, such as beadslurries, for example. Although progress has been made, there is a needfor improved diaphragm construction of microassay cartridges, and inparticular for a process applicable to miniaturized circuit elements.The present invention provides these and related advantages.

BRIEF SUMMARY

To improve the efficiency and speed of operation of a microfluidiccircuit element such as a valve or a pump, it is desirable that the workrequired to change from a first state to a second state is minimal. Apreferred class of circuit elements is diaphragm operated. Thepneumatically controlled diaphragm separates a pneumatic subcavity froma hydraulic subcavity and operates on a fluid contained in the hydraulicsubcavity. The diaphragm “web” is a thin film that serves as a barrierbetween the two subcavities, dynamically translating pneumatic pressureinto fluid motion, or stasis. In a first state, the diaphragm web is ina first position between the subcavities, in a second state thediaphragm web is displaced from the first position and occupies a secondposition. Generally the first position conforms proximately to aninterior surface of the hydraulic subcavity and the second positionconforms proximately to a second interior surface of the pneumaticsubcavity, and by exerting a force, the diaphragm may be reversiblytransitioned between the two positions or states.

Unfortunately, currently existing microfluidic diaphragms are generallyelastomeric in nature and require overcoming the significant elastomericresistance to change from a first state to a second state. Therefore, itis desirable if the work required to change from one state to the otherwas reduced. We have invented a novel barrier that has a substantiallyzero work function to change from the first state to the second state.This is accomplished by the use of a web having near zero elastomericproperties and a surface area significantly larger than the microcavityin to which the web is sealed. Most preferably, the surface area of thediaphragm web closely approximates the interior surface area of asubcavity, or if the subcavities are not symmetrical, then the surfacearea of the web closely approximates the interior surface area of thelarger of the subcavities. The diaphragm web is thus a movable filmhaving low elasticity for separating a hydraulic and a pneumaticsubcavity of a microfluidic circuit element, where the area of the filmis larger than the greatest cross sectional area of the microcavity.

Film Properties

It is desirable for the web for use in these microfluidic cartridges bea film that is not significantly elastomeric and generally matches theinterior surface area of one subcavity the target microcavity,preferably the interior surface area of the larger subcavity of themicrocavity. Most preferably, the film requires little or no work totransition from one state to another. The film is desirably in a flaccidstate until the applied control pressure drives the film to one side orthe other of a microfluidic cavity by inverting the film position.Preferably, the film can also be described as having near zero or zerorestorative force toward a reduced area state. Most preferably, the filmis a low or non-elastomeric film that does not significantlyself-restore to a form with a surface area approximating thecross-sectional area of the cavity, and substantially matches theinterior surface area of the subcavities without significant over orunder pressure. Using a non-elastomeric film with a surface areamatching the interior surface enables several advantageous and novelproperties.

The use of low or non-elastomeric films as microfluidic componentsenables the production of valves, pumps and microfluidic features thathave advantageous features. Notably through the use of low ornon-elastomeric films, the restoring force of an elastomeric film doesnot need to be overcome as the film is moved from one side of themicrocavity to the other. This reduction in force arises because thepneumatic control needs only overcome the inertia of the membrane andfluid, not the elastic spring force of the film plus the inertia of themembrane and fluid. This enables a faster cycling and/or cycling withreduced pressure, or both, of the membrane between one side and theother, e.g. from the open state to the closed state for a valve.

Manufacture

The yielded film in a microfluidic assembly can be formed by a number ofmethods. The films can be formed after assembly of the microfluidiccartridge by applying sufficient pressure to stretch the film over itsyield point, or with a mechanical press for stretching the film into acavity in the microfluidic assembly, or they can be stretched by the useof a punch and die prior to assembly, such as by a process ofpre-stretching diaphragm webs in bulk. Depending upon the film and themanufacturing processes it may be desirable to form the films with oneor more of these processes. After typical manufacture, some films areflexible but have substantially no elasticity in their range of motion.By the use of suitable dies and forming processes these films can beformed into shapes complimentary to the target microfluidic cavities.For example it is known in the art that a heated vacuum die can be usedstretch films into macroscopic bubbles having a generally cylindricalshape. By the use of a suitable die, the films may be stretched on amicrofluidic die before the film is aligned with the first microfluidicassembly and then continuously bonded to said first microfluidicassembly. Through the use of continuous roll to roll processes,significant time and cost savings in manufacture may be realized.

To generate a film by a yield in place process, the surface area of theassembled film and the area of at least one portion of the microcavitybe of a ratio that when a pressure difference is applied to the chip,the film is forced to match the interior surface area of the cavity andstretch the film beyond its yield point. This process may beaccomplished during the cartridge assembly, or after assembly, when itmay be yielded as part of the manufacture process, or by the initialoperation of the microfluidic cartridge. Some films such as SARANEX® canbe yielded in place with a relatively low pressure difference. To yieldother films, it may be necessary to provide additional externalpressures to ensure that the desired yield pressure does not cause amechanical failure of the microfluidic assembly.

The films can also be yielded in place during manufacture. This may beaccomplished through the pressure differential method described above.Alternatively, the yielding can arise through mechanical means. Forexample, a punch and a partially assembled microfluidic cartridge can beused. The film can be bonded to one side of the microfluidic assembly.After bonding the film, a die can be mechanically pressed into the film,driving the film into the void below. Alternatively, the film may beexpanded into a die, and the expanded film is then transferred intomicrocavity void. Through the choice of a suitable die and pressure, thefilm can be stretched into a non-elastomeric state. For some films itmay be desirable to perform the mechanical stretching at elevatedtemperatures. For some choices of film and manufacturing speeds, it maybe desirable to form the yielded films prior to the assembly step. Thiscan be accomplished through the use of a suitable molds or dies to formthe complimentary pattern of stretched films in the larger carrier film.It may be advantageous to cut the resultant stretched film portions fromthe carrier film upon transfer to the microfluidic subassembly. This canbe done either by kiss cutting with a die, or by a selective cuttingwith a laser film cutter.

The film can also be yielded prior to assembly. In this case, theassembly process needs to gather sufficient non-elastomeric film to linethe surface area of the desired target cavity. This can be accomplishedthrough the use of a punch and die combination, or by vacuum or pressureforming the film into a die, wherein the die has dimensions similar tothe target microcavity. The film can then be positioned on themicrofluidic subassembly and the non-elastomeric film transferred by asuitable change in pressure. Those skilled in the art can recognize thatit may be advantageous to insert manufacturing steps in the process,such as transferring the film and a perimeter to an adhesive layer,bonding the ungathered film with heat, pressure, or solvent, applyingadhesive to the ungathered material, cutting a perimeter to create abondable area for the yielded film, or combinations thereof. When thefilm is yielded prior to cartridge assembly, manufacturing conditionsthat might be harmful to the microfluidic chip may be used.Specifically, it may be desirable for manufacturing reasons to useeither pressure or thermal processing steps that may be incompatiblewith the microfluidic assembly, or reagents therein. By performing theyield process off of the microfluidic assembly, it becomes possible touse combinations of pressure and temperature that are relativelyinaccessible to an assembled chip. This permits the use of polymers suchas polyimide that have optimal process conditions that exceed thestrength, and or desired temperatures desirable for plastic microfluidiccartridges. By utilizing these manufacturing techniques, those skilledin the art will appreciate greater flexibility microfluidic cartridgedesign and manufacture.

In all cases, it is desirable that the film be stretched sufficiently toirreversibly yield the material. Specifically, the stretch appliedshould exceed the yield point of the film to create non-elastomeric orvery low elasticity film such that the yielded film has a surface areaand shape complimentary to the inner surface of the microfluidic cavityinto which it is assembled. It is known in the art that not allmicrofluidic cavities are symmetric with respect to the film layer, andin these cases, it may desirable that the surface area of the film matchthe larger of the two microcavities for most uses. For some uses, it maybe sufficient to have the film match the surface area of the hydraulicor pneumatic side only. For example, it may be desirable with severalmicrocavities in sequence to have the operating volume change bedifferent amongst the microcavities. This can be readily accomplished byhaving an asymmetric division of the microcavity by the film.

Diaphragms of the invention form components of micropumps, microvalves,and microvents. A micropump is one such inventive combination, themicropump comprising a cavity having a first subcavity configured toreceive a fluid; a second subcavity configured to be reversiblypressurized; a diaphragm interposed between and separating the firstsubcavity from the second subcavity; and, wherein the diaphragm is apolymeric thin film web having a yield point and is characterized by apermanently overstretched deformation of the web. The thin film webinelastically conforms in a first state to a first internal surface ofthe micropump cavity when pressurized and in a second state to a secondinternal surface of the cavity when depressurized. The micropump isconfigured to pump a liquid according to a pump stroke defined by thereversible motion of the permanently overstretched deformation of theweb between the first state and the second state as driven bypressurization and depressurization of the second subcavity.

A second combination is a microvalve, the microvalve comprising a cavityhaving a first subcavity configured with a valve inlet, a valve outlet,and a valve seat interposed between the valve inlet and the valveoutlet, wherein the first subcavity is configured to receive a fluid; asecond subcavity configured to be reversibly pressurized; a diaphragminterposed between and separating the first subcavity from the secondsubcavity, wherein the diaphragm is enabled to be reversibly deflectedagainst the valve seat according to whether the second subcavity ispressurized or depressurized, thereby defining an “ON” position and an“OFF” position of the microvalve; and, further characterized in that thediaphragm is a polymeric thin film web having a yield point and ischaracterized by a permanently overstretched deformation of the web. Thethin film web inelastically conforms in a first state to a firstinternal surface of the cavity when pressurized and in a second state toa second internal surface of the cavity when depressurized. Themicrovalve is configured to open and close by the reversible motion ofthe permanently overstretched deformation of the web between the firststate and the second state as driven by pressurization anddepressurization of the second subcavity. The invention also comprisescombinations of the diaphragm elements as components of microfluidiccircuits and devices.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B are renderings of a diaphragm member being stretched bya pressure applied within a sealed chamber. Whereas elasticallystretchable materials return to a relaxed state, inelastically stretchedmaterials undergo permanent deformation.

FIGS. 2A and 2B illustrate schematically two states of an inelasticallystretched material that undergoes collapse when depressurized.

FIG. 3 depicts a stretched diaphragm film conforming to the interiorwall of a concave chamber.

FIGS. 4A and 4B depict a chamber in cutaway view, the chamber havinggenerally vertical walls and layered construction. Illustrated is thegeometry of a diaphragm web in process of being stretched to conform tothe chamber.

FIGS. 5A and 5B are perspective views of a cutaway section through agenerally rectilinear chamber, showing progressive stretching of thediaphragm web to conform to the interior surface area of the upperchamber subcavity.

FIGS. 6A, 6B, and 6C are plan and elevation views of a stretcheddiaphragm member. FIGS. 6B and 6C depict a first state and a secondstate, where the first state is distended and the second state iscontracted.

FIG. 7 is an exploded view of a diaphragm assembly in a chamberconstructed by lamination. A diaphragm element having an apron is shownwithin the stack of layers.

FIG. 8 is an exploded view of a layered chamber where the diaphragmelement is formed from a layer of the device.

FIGS. 9A and 9B illustrate the operation of a diaphragm film.

FIGS. 10A and 10B are plots of stroke volume before and after a cycle ofstretching of a film past its elastic limit.

FIG. 11 is a stress-strain analysis for a co-laminate film of lowdensity polyethylene/ethylene vinyl acetate/polyvinylidenechloride,/ethylene vinyl acetate, and low density polyethylene.

FIG. 12 is a stress-strain analysis for a film of an elastomericpolyurethane.

FIG. 13 is a stress-strain analysis for a film of a high modulusmaterial: polyethylene terephthalate.

FIG. 14 is a stress-strain analysis showing hysteresis in three serialstretch and relax cycles of a co-laminate film of low densitypolyethylene/ethylene vinyl acetate/polyvinylidene chloride,/ethylenevinyl acetate, and low density polyethylene

FIG. 15 is a stress-strain analysis showing hysteresis in three serialstretch and relax cycles for polyethylene terephthalate.

FIGS. 16A and 16B are cross-sectional views of a microvalve structure,showing an “ON” and an “OFF” configuration of the valve diaphragm. Thevalve diaphragm member is formed by a process of inelastic deformation.

FIG. 17 is a cutaway view of a valve with inelastically deformeddiaphragm.

FIGS. 18A and 18B are plan and elevation views of an overstretcheddiaphragm member for a fluidic microvalve.

FIG. 19 is an exploded view of a valve structure having an overstretcheddiaphragm.

FIGS. 20A and 20B are cross-sectional views of a microvalve structure,showing an “OPEN” and an “OFF” configuration of the valve diaphragm.

FIG. 21 is a perspective view of a diaphragm web of FIG. 20.

FIG. 22A illustrates the concept of valve latency. Data for apre-stretched yield-in-place valve is compared to a conventionalelastomeric valve in FIG. 22B. The latency time is reported inmilliseconds.

FIG. 23 is a theoretical analysis of the relationship between a criticalweb dimension L_(c) and a process pressure required to achieve a definedoverstretch.

FIG. 24 is an experimental study of overstretch behavior in a diaphragmvalve.

FIGS. 25A through 25F are sequential views of steps in a first processof mechanically stretching a diaphragm film in place.

FIGS. 26A through 26F are sequential views of steps in a second processof mechanically stretching a diaphragm film in place.

FIGS. 27A and 27B illustrate a close-ended channel with breathablediaphragm for fluid loading and priming of a pump having a breathablediaphragm.

FIG. 28 is an exploded view of a close-ended channel with breathablediaphragm having a body constructed of layers.

FIGS. 29A and 29B are schematic views of a flow-through fluidic elementwith microporous film for gassing or degassing a liquid.

FIGS. 30A, B and C are electron micrographs of the fine structure of abreathable microporous polyurethane film.

FIG. 31 illustrates a representative fluidic circuit having acombination of diaphragm-operated circuit elements of the invention.

FIG. 32 illustrates a microfluidic cartridge formed of pneumatic andhydraulic circuits containing microvalves and micropumps of theinvention.

FIG. 33 tabulates parameters such as yield stress of high and lowmodulus films and is useful in guiding selection of diaphragm materialsfor yield-in-place valve applications.

DETAILED DESCRIPTION

Although the following detailed description contains specific detailsfor the purposes of illustration, one of skill in the art willappreciate that many variations and alterations to the following detailsare within the scope of the claimed invention. The following definitionsare set forth as an aid in explaining the invention as claimed.

DEFINITIONS

A “cartridge” is an analytical device designed for operation byinsertion into a host instrument. The host instrument supplies thepneumatic pressure, pulses, and detection means for performance of theassay. The cartridge contains hydraulic works and pneumatic works,including microscale channels, cavities and chambers. Sample and reagentliquids are conveyed in a hydraulic network of the cartridge or card;fluid flow is controlled and driven by a pneumatic network thatinterfaces with the hydraulics at diaphragms spanning selectedjunctions, channels and chambers. Typically, the body of the cartridgeor card is made of a flexible plastic and may be formed by lamination,molding or a combination thereof. Body plastics may include, but are notlimited to, polycarbonate, polyethylene terephthalate, cyclicpolyolefins, acrylates, methacrylates, polystyrene, polyimide,polysilicone, polypropylene, high density polyethylene, low densitypolyethylene, graft and block copolymers, and composites thereof. Apreferred cartridge is made from rollstock and includes dry reagentsprinted thereon. Other such cartridges may include molded body elements.

“Hydraulic works” of a device: includes the network or networks ofintercommunicating channels and chambers that are intended to be wettedby sample or liquid reagents in the course of an assay. The hydraulicnetworks are configured with fluidic subcircuits for performing thesteps of an assay.

“Pneumatic works” of a device: includes the network or networks ofpneumatically actuated valves, pumps and diaphragms and interconnectingcircuitry and manifolds that are useful for powering and controlling thehydraulics of the device. The pneumatic works of the cartridge deviceinterface with positive and negative pressure sources on the hostinstrument and with valves, diaphragms, pumps and other pneumaticallyactuated elements that control and drive liquids in the hydraulicnetwork.

While it may be said that the pneumatic works of the device arepreferably operated with a gas such as air or nitrogen, it is alsoconceived that equivalent “pneumatic” circuits may be operated with afluid more generally, where fluid is selected from a gas or a liquid,including liquids such as silicone oils, vegetable oils, fluorocarbonliquids, and the like. Thus in one variant of the invention, thepneumatic works are operated with a “fluid” having the characteristicsof a liquid and the operation of the device is otherwise equivalent, aswould readily be understood by one skilled in the art.

“Fluidic works” of a device: include the hydraulic works formed of anetwork or networks of internal channels and chambers wetted in thecourse of the assay and the pneumatic works formed of control and pumpdriving circuits powered by positive and negative pressure sourcesderived from a host instrument via a pneumatic interface.

The fluidic works may be divided into fluidic subcircuits, where eachsubcircuit comprises channels and chambers for performing a particularfunction on a liquid sample or reagent. The fluidic subcircuits may beorganized into serial subcircuits (such as for extraction, amplificationand detection of a nucleic acid target or targets) and parallelsubcircuits and networks such as for simultaneous assay for multipletargets on a single sample by splitting the sample. “Microscale” and“fluidic” refer to devices having submillimeter features.

“Microfluidic”—by convention, refers to fluidic features having at leastone critical dimension that is generally less than 500 micrometers. Thenarrowness of the critical dimension results in fundamental changes inthe rules governing fluid flow. The liquid flow regime is characterizedby Poiseuille or “laminar” flow.

“Stress” is the internal or restoring force per unit area associatedwith a strain and has units of Pascals or megaPascals.

“Strain” is a ratio ΔL/L₀ of the change in length divided by theoriginal length in response to an externally applied stress, and isunitless; it is often given in percent.

“Yield point” is the point on a stress-strain curve where the curvedeflects or levels off and plastic deformation commences, and thuscorresponds to the “elastic limit” of the material. Prior to the yieldpoint, the material elastically return to its original shape when theapplied stress is removed. Once the yield point is passed, some fractionof the deformation will be permanent and non-reversible. A yieldedmaterial, such as a diaphragm, has been stretched beyond its yieldpoint.

“Yield Strength” and “yield point” are measured by standard techniquesfor reproducibility, such as described in ASTM Test Method D882-10 (the“882 test method”). For consistency, generally a 1 mil film is apreferred substrate. Yield strength is an indication of the maximumstress that can be developed in a material without causing irreversibledeformation. Yield point is an indication of the maximum strain that canbe developed in a material without causing irreversible deformation. Forpractical reasons, the measurements of yield strength, strain, elasticlimit and elastic modulus are defined experimentally from astress-strain diagram.

Offset yield strength is the stress read from the plot at theintersection of an offset line (drawn parallel to the initial slope ofthe stress-strain curve through the elastic deformation range) and thestress-strain curve, where the offset line is offset by a selectedvalue. Offsets for plastics are conventionally taken as 2%. Optionally,yield is sometimes shown as a range, for example in the case ofco-laminated films.

“Elasticity” refers to the ability of a material to return to itsoriginal shape when load causing deformation is removed. Elasticity isthe ability to store and release energy with a spring-like sampleresponse generally as described by Hook's law of elasticity. If thestrain increases linearly with increasing applied stress, the materialis purely elastic, however in some materials, such as materialsdisplaying viscoelastic properties, the stress-strain relation is notlinear and the sample response is strongly dependent on time and rate ofload application.

“Elastic modulus” (E), also termed “Elastic Modulus”, is a slopemeasured in the elastic deformation region of the stress-strain curve,where strain is fully reversible. “Elastic Modulus” is the initial slopemeasured in the stress-strain curve and is an indication of thestiffness of the material. Elastic Modulus is a constant within therange of stretch or deformation that is fully reversible, and is thusequivalent to the spring constant of Hooke's Law.

“Permanent Deformation” or “inelastic deformation”, is an increase inlength dimension, expressed as a percentage of the original lengthdimension, by which material fails to return to its original lengthafter being subjected to an elongation stress. When subjected to astress greater than the yield strength or elastic limit of the film,permanent deformations of thin films may occur. For example, when a thinfilm diaphragm having a web span length from one side to another of acavity or frame is stretched by a pressure and then collapsed back to arelaxed state, the web span length may be permanently elongatedaccording to the amount of overstretch that the diaphragm was subjectedto in excess of its yield point. “Overstretch” simply indicates that thematerial has been stretched past its yield point.

“Toughness” of a material is the ability of a material to absorb energyand plastically deform without fracturing or rupturing, and can berelated to the total area under the stress-strain curve up to a breakpoint according to the integral

K=∫ ₀ ^(ε) ^(f) σdε

where ε is strain, ε_(f) is the strain on failure, and σ is stress. Theunits of K are of energy per unit volume. For purposes of the invention,toughness is particularly indicative of the capacity of a material toundergo a strain of up to 50% by length and to be permanently deformedthereby. This property is desirable for the manufacture of pneumaticelements by a form-in-place process as described herein.

A comparison of the relative magnitudes of the yield strength, ultimatetensile strength and percent elongation of different material can alsogive a good indication of their relative toughness.

“Top”, “bottom”, “up”, “down”, “upper”, “lower”, “above”, “below”,“upward”, “downward”, “superior to”, “floor”, “roof”, “convex”,“concave”, and so forth, are indications of relative position and notabsolute position or viewpoint: when reference is made to a specificframe of reference, such as the “ground plane”, as taken orthogonally toan intersecting plumb line.

Unless the context requires otherwise, throughout the specification andclaims that follow, the word “comprise” and variations thereof, such as,“comprises” and “comprising” are to be construed in an open, inclusivesense, that is as “including, but not limited to”. Reference throughoutthis specification to “one embodiment”, “an embodiment”, “one aspect”,or “an aspect” means that a particular feature, structure orcharacteristic described in connection with the embodiment or aspect maybe included one embodiment but not necessarily all embodiments of theinvention. Furthermore, the features, structures, or characteristics ofthe invention disclosed here may be combined in any suitable manner inone or more embodiments. “Conventional” is a term designating that whichis known in the prior art to which this invention relates. “About” and“generally” are broadening expressions of inexactitude, describing acondition of being “more or less”, “approximately”, or “almost” in thesense of “just about”, where variation would be insignificant, obvious,or of equivalent utility or function, and further indicating theexistence of obvious minor exceptions to a norm, rule or limit.

Microfluidic Circuit Elements by a Yield-in-Place Process

Conventional art relies on diaphragms formed from elastomeric films orrigid sheets. However, we have found surprisingly that a hithertounrealized class of diaphragm materials may be advantageously used informing microvalves and micropumps. These diaphragm materials arepolymers selected for toughness and chemical resistance, butsubstantially lack elasticity once stretched and are not excessivelystiff. The unifying concept is a recognition that an overstretchedmaterial (e.g., yielded) selected for its toughness, having beenstretched beyond its yield point so as to be deformed permanently,requires essentially no work to transition from a first state to asecond state, each state conforming to an opposite aspect of amicrocavity. The diaphragm is typically stretched before use or on firstuse and behaves as a flexible, flaccid, blister-shaped skin,inelastically controlling or propelling fluid flow according to thepressure differential across the diaphragm. Once stretched, thediaphragm film does not return to its native dimensions, andadvantageously, this results in decreased latency and increased strokevolume, decreased incidence of sticking of the diaphragm to the pumpwall, and improved conveyance of bead slurries, for example.

FIGS. 1A and 1B show a plastic film diaphragm member being stretched bya form-in-place process of “over-stretching” the film beyond its yieldpoint. In this case, a simplified pneumatic manifold 2500 and diaphragm2501 is shown. The edge of the diaphragm is affixed to the substrate2502 and an apron 2503 extends past the periphery of a pneumatic cavity2504. The diaphragm itself forms a “web” over the cavity. Duringstretching under controlled process conditions, the diaphragm webcovering the lower chamber is stretched and permanently deformed asshown in FIG. 1B. A fluid entering portal 2505 expands the diaphragmlike a soap bubble, but upon release of pressure, instead of relaxing toits original sheet-like state, the diaphragm web will remain permanentlystretched, forming a collapsible, flaccid, blister-shaped skin, and willhave a larger stroke volume and decreased work function in transitioningfrom a distended state to a collapsed state. In contrast, an elasticmaterial will resist distension and will recover to its native flatnesswhen the pressure is removed.

For micropump and valve diaphragms formed by a yield-in-place process,materials having a yield point (when tested by ASTM D882 standardmethods) in the range of about 2 to 30 MPa are preferred, but materialshaving a yield point of about 100 or 120 MPa may be used if desired.Diaphragm materials having yield points of 30 MPa or less include, forexample polyethylene, low density polyethylene, blends with high densitypolyethylene, polyolefin composites and laminates, polyvinylidenechloride composites and laminates, and ethylene vinyl acetatecomposites, while not limited thereto. It is well known that blends,block grafts and co-laminates (generally termed “composites”) of thesepolymers may be formulated to tailor the yield strength for a particularapplication, for example a co-laminate consisting of low densitypolyethylene/ethylene vinyl acetate/polyvinylidene chloride,/ethylenevinyl acetate, and low density polyethylene (LDPE/EVA/PVDC/EVA/LDPE) (assold under the trade name SARANEX®) was found to have a yield strengthof about 15 MPa and has been demonstrated to be useful in the inventivemicropumps and microvalves. Typically the films employed will have athickness of 1 to 3 mils, although slightly thinner or thicker films mayalso be used. Other useful films will be apparent to one skilled in theart after study of this disclosure. One useful film having a yield pointof about 100 MPa is polyethylene terephthalate, which is readilyavailable in a 1 mil thickness in sheets or rolls, and which may beformed into stretched diaphragm webs of the invention by mechanicalmeans as will be described below.

FIGS. 2A and 2B are views of a diaphragm film 2501 in a “distendedstate” and a “collapsed state”. In the distended state (FIG. 2A) thefilm is essentially concave in shape, and in the collapsed shape (FIG.2B), the film is generally amorphous and can be pressed flat. We havefound, surprisingly, that the films function well as microvalve andmicropump diaphragms. When pressed flat against a valve seat, forexample, liquid flow through a valve is readily stopped under operatingconditions typical for a microfluidic device. When operated as a pump,the pump stroke is essentially equal to the volume enclosed in thedistended state less the volume enclosed when pressed flat by a pressureor a mechanical actuator, i.e., the micropump has essentially a zerodeadspace volume.

In FIG. 2B, the amorphous web is collapsed but has not recoveredelastically to its native flatness, and now has a surface area that issubstantially greater than its virgin condition, having beenoverstretched past its yield point. The figure is drawn schematicallyand is not intended to represent the actual appearance of a distended orcollapsed diaphragm member in use. Stretched diaphragm webs of this typemay be formed with a surrounding apron 2503 as shown here, where theapron serves as lip to attach and seal the diaphragm to the substrate.

FIG. 3 is a schematic view defining the analytical geometry ofmechanical strain in a diaphragm member adapted for a subcavity shapedas a spherical cap 2510. In this example, the cavity is defined by aspan dimension or chordal length L from A to B and an internal heightequal to the sagitta or height of the spherical cap segment. Thediaphragm 2501 conforms to the interior surface of the subcavity 2510.Here the cap height is denoted by z, and is a fraction of the radius R.The internal wall of the subcavity from A to B defines an arc length L′of a sphere 2511 having radius R, where the arc is inscribed by centerangle θ, and where O is the center of the sphere. Pump cavities of thiskind can be made with a hemispherical milling head or by a moldingprocess, for example. The arc length L′ is then calculated from thecentral angle θ (in radians) and the radius R by the formulae L′=θ*R sothat the overstretch can again be calculated as a permanent deformationL′/L. The permanent deformation, when expressed as a percentage, may becompared to a stress-strain curve for the material, where it will beseen that the deformation of the diaphragm webs of the invention exceedsthe elastic limit of the material on the strain axis.

FIG. 4A is a cross-sectional view of a rectilinear pump microcavity 2520formed by lamination, the laminate stack including a sheet of diaphragmmaterial 2501. In this example, the diaphragm is a continuous sheet andextends throughout the device body. The diaphragm layer separates thedevice into a lower body part and an upper body part. The lower bodypart includes hydraulic subcavity 2522, inlet 2523 and outlet 2524. Theupper body part includes a pneumatic subcavity 2525 and pneumaticactuation port 2526. In this example, the two body parts are joined by aglue layer 2527. The structure may be assembled layer by layer, or maybe assembled by first forming a lower body part, applying the diaphragmlayer 2501 to the body part, and then building or adding an upper bodypart thereon. As shown here, the distended state “blister” may be formedby a yield-in-place process after the body has been assembled, generallyby applying a pressure exceeding the yield point of the thin filmmaterial but not exceeding the mechanical strength of the cartridgebody. Optionally, the blister may instead be formed before the finalassembly is completed.

FIG. 4B is a schematic view defining the analytical geometry ofmechanical strain of a diaphragm member adapted for a rectilinear cavity2530. The cavity is characterized by a span dimension from A to B and aninternal height h. Diaphragm 2531 (dashed lines) forms a “web” spanningand sealing the cavity. The dimensions are not necessarily constant onany axis, but for simplicity of explanation it can be seen that theinternal surface of this rectilinear cavity is bounded by a lengthL′=2h+L in this sectional view, where L is the length from A to B and his the interior height of the cavity. When yielded under an appliedpressure, the diaphragm web 2531 will stretch to generally conform tothe internal dimensions of the cavity and will acquire a length L′≈2h+L,where the length L′ is generally equal to two times the height of thechamber plus the web span length from side to side. The overstretch(L′>L) can then be quantitated and equals a permanent stretchdeformation factor of L′/L. To achieve this, materials are selected sothat the yield point (elastic limit) of the material is exceeded in thestretching process. Stretching is not necessarily uniform, and in somecases will be greatest along the peripheral border of the web.

FIG. 5A is a cutaway view of a pump structure 2540, depicting amicrofluidic device with diaphragm 2541 in an overstretchedconfiguration which partially fills the cavity. In FIG. 5B the diaphragmis stretched to more closely conform to the interior walls of thecavity. The diaphragm is advantageously and simply made by forming thepump structure to include a flat sheet of the selected diaphragm filmsandwiched in place; then in a separate process step, the device issubjected to an internal pressure on one side of the diaphragm, wherethe pressure is configured to exceed the yield strength of the diaphragmfilm, thus irreversibly stretching the film. We term this a“yield-in-place” process. The diaphragm in its distended state afterstretching is seen to have a convex “blistered” or “ballooned”appearance that can be readily collapsed as earlier described withreference to FIG. 2B. Also shown in the figure is an “apron” 2542 usedto seal the diaphragm between the pneumatic housing members (upperlayers) and the fluidic housing members (lower layers). The apron inthis example is contacted by a glue layer 2543 that bonds the fluidicface to the pneumatic face of the device. Also shown are pump inlet 2544and outlet 2545, where directionality of fluid flow is established forexample by the use of check valves (not shown) or other means known inthe art. Also shown is pneumatic actuator port 2546 and pneumatic cavity2547.

FIGS. 6A, 6B and 6C are a plan view and elevation views of ayield-in-place diaphragm member 2541 for a fluidic circuit element. Inplan view, the apron 2542 is seen to be configured to seat in agenerally cylindrical chamber. View 6B shows the appearance of theuncollapsed blister after its manufacturing process is completed insitu; however it will be understood that upon release of pressure, theweb blister generally will collapse into an amorphous folded form and/orcan be pressed so as to flatten against an opposing surface of thesubstrate, as drawn conceptually in FIG. 6C. The web has two states, afirst distended state as shown in FIG. 6B and a second state as shown inFIG. 6C. However, it is understood that the film may be equallydistended in both states, but at opposite sides of a microcavity, aswould be the case if the microcavity consisted of hydraulic andpneumatic subcavities that had generally mirror symmetry. The two statesmay be described as being endpoints of a transitional process in whichthe diaphragm moves or inverts from the first state to the second state.In some instances, the pressure difference required to invert the filmfrom a first state to a second state within the cavity is less than 3psi. In other instances, the pressure difference required to invert thefilm within the cavity is less than 1 psi. In yet another workingexample, the pressure difference required to invert the film within thecavity is less than 0.1 psi and is substantially less than the pressuredifference required to overcome the inertia of the liquid in thehydraulic cavity.

FIG. 7 is an exploded view showing the construction of a pump structure2540 by a process of lamination and includes a glue layer 2543. In thisexample, the blister shape of the diaphragm member 2541 is made afterassembly by a “form in-place” process in which the yield strength of thediaphragm web is exceeded by an applied pressure. The diaphragm will bestretched to conform to the internal surface of the pneumatic cavity2547. The diaphragm member has been cut to fit so that a single gluelayer may be used.

FIG. 8 is an exploded view of a pump structure 2550 formed bylamination, where an uncut sheet of diaphragm material 2551 is layeredin the assembly stack. Two glue layers (2552 a, 2552 b) bond thediaphragm layer in the stack. The blister shape is again formed afterassembly by a process of applying an internal pressure on one side ofthe diaphragm. The diaphragm will be stretched to conform to theinternal surface of the cavity.

FIGS. 9A and 9B demonstrate how a diaphragm-driven micropump 2560 can beformed by a yield-in-place process of yielding a plastic film. Thedifference in enclosed volume between the stretched diaphragm web (2561,FIG. 9B) and base 2563 a of the hydraulic subcavity 2563 is the ejectionstroke volume of the pump. At full stretch, the diaphragm web conformsproximately to the interior roof and walls 2562 a of the pneumaticsubcavity 2562. Thus the process of manufacturing mimics the process ofpumping and ensures a consistent stroke volume. This was first notedwith films that were not resilient elastomers but had been chosen forchemical resistance. A particular film in this class was a co-laminateof a polyethylene terephthalate or vinylidene chloride sandwich disposedbetween layers of polyethylene, which is valued for its chemicalresistance. We found that the first ejection stroke volume of a virginfilm was significantly less than second or third ejection stroke volumeof the film under normal conditions of use. After investigation, it wasdiscovered that the film had stretched past its yield point and wasirreversibly deformed by the process. In use, the film is stretched soas to approximately conform and have the surface area of the interiorwall surface 2562 a of the pneumatic subcavity 2562. Also shown in thefigure are the hydraulic cavity 2563, liquid inlet 2564, liquid outlet2565, glue layer 2566, molded substrate layer 2567, and pneumaticactuation duct 2568.

The form-in-place process is advantageous in its simplicity, but othermeans for forming an overstretched diaphragm include use of male and/orfemale molds to form the “blister” features on the sheet prior toassembly, where a sheet having pre-formed blisters is mated to align thelocally-stretched diaphragm features with pre-formed cavities in thecartridge body. Pre-stretching of the diaphragms may be done with amechanical platen press, or can involve a roll-to-roll process using arotating die. Vacuum forming of the stretched web elements is alsoconceived.

In another variant of the process, a sheet of a thin film material maybe layered over a pre-formed body half having cavities and circuitfeatures. A press or a soft roller may then be used to stretch the filminto the cavities, and a second body half may then be mated with thefirst to sandwich the diaphragm features in place. Excess material maybe removed if desired. In some instances the diaphragm and the cartridgebody are made of like or similar materials and can be bonded by thermal,ultrasonic or solvent welding. In other instances glue is used.

FIGS. 9A and 9B also demonstrate a comparison of thin film diaphragm2561 in a virgin versus an overstretched configuration. In theoverstretched configuration, the distended web of film 2561 has anarcuate length measured along its surface from edge to edge of thecavity 2505 that is longer than the chordal length (as measured alongthe unstretched film). The surface area of the stretched web memberapproximates the surface area of the internal roof and walls of thepneumatic subcavity 2562 (or will have the surface area of the larger ofthe two subcavities when stretched to fit). The enclosed volume of thestretched state is greater than the collapsed or relaxed state. Thusoperating the device by use of pneumatic pressure at pressure port 2568can be used to drive the diaphragm from a distended to a collapsed statein alternation, thus achieving a pump stroke for filling and ejectingfluid from subcavity 2563. These pumps are self-priming.

Stroke volume maturation is shown in FIGS. 10A and 10B. Ejection strokevolume of a stretchable plastic film before (PRE) and post (POST)stretch past the yield point is shown to result in a gain in strokevolume. It was discovered that diaphragm webs were stretching afterassembly because the stroke volume increased with repeated use; thisphenomenon was then exploited to form pre-stretched diaphragms having amaximal stroke volume for the dimensions of the pump cavity.

As a matter of quality control and reproducibility of operation, itproved advantageous to perform this stretching process prior to releaseof product or to conduct a “warm up” operation prior to starting anassay. Once the stretching process is complete, the stretched diaphragmsoperate with an increased ejection stroke volume (and decreased responsetime) that is no longer dampened by the elasticity of the film, as hadbeen problematic with pumps and valves of the prior art. Materials maybe used that are tougher and more chemically resistant than thepolyurethane rubber diaphragms of the prior art. Typically thesematerials have yield points under 30 MPa, and more preferably under 20MPa for micro-dimensioned fluidic features, but the blister may also beformed using mechanical means as described below, thus allowing thoseskilled in the art to form the inventive yielded webs from materialshaving higher yield strengths and correspondingly higher elastic moduli.Once stretched, the resistance required to transition the diaphragm webfrom a distended to a flaccidly collapsed state is negligible, such thatthe work required to move the film is essentially only that needed toovercome inertia of a fluid in the chamber, with no added work requiredto overcome the restorative force of the elastomeric diaphragms of theprior art.

As shown in FIG. 10A, ejection stroke volume for a SARANEX® diaphragmhaving a diameter of about 1.08 cm was found to increase from about 90microliters (PRE) to 150 microliters (POST) by becoming overstretched,more than a 50% increase. The cavity ceiling (FIG. 9B, 320) limitsultimate stretch dimensions of the film and ensures a higher level ofconsistency of the nominal stroke volume in the manufactured product.

Similarly, as shown in FIG. 10B, a diaphragm having a diameter of about0.88 cm was found have an ejection stroke volume of 50 microliters (PRE)before stretching and about 90 microliters (POST) after stretching,about an 80% increase. Diaphragms which have been stretched may bestored in a collapsed state until needed.

To better understand the materials behavior underlying the results shownin FIG. 10, we performed stress-strain analyses of severalrepresentative films. Yield stress and yield strain (load anddeflection) were measured on an Instron universal materials testingmachine (Instron, Norwood, Mass.) operated at strain ramping speed of150 millimeters/min, a gauge length of 15 cm between grips, and a gripwidth of about 2.2 cm. Samples of films were generally 1 mil inthickness unless otherwise noted. Yield strengths were determined bystandard methods as outlined in ASTM Test Protocol D882 (100 Barr HarborDr., PO Box C700, West Conshohocken, Pa. 19428). Testing is generallydone at a controlled room temperature of about 23° C. Measured yieldstrengths are dependent on strain rate, temperature, and filmcharacteristics, and one skilled in the art will understand that thematerial property parameters cited here are given with reference tostandardized testing conditions.

One material of interest is low density polyethylene/ethylene vinylacetate/polyvinylidene chloride,/ethylene vinyl acetate, and low densitypolyethylene (LDPE/EVA/PVDC/EVA/LDPE) co-laminate, sold under the tradename SARANEX®. FIG. 11 is a stress-strain analysis for a PVDC/PET/PEco-laminate film. The material has a limited elastic modulus and beginsto deform at a yield point in the range of 12-16 MPa. The slope over thereversibly elastic range corresponding to an elastic modulus E=˜80 MPa.At 50% on the strain axis, the material was allowed to relax, butdisplays an intermediate level of hysteresis with a return to zerostress, indicating a permanent deformation of 30% (i.e., L′/L). Thisoverstretch results in an essentially stress-less response in subsequentcycles, as shown below.

FIG. 12 depicts a stress-strain curve obtained with a 1 mil film ofPolyurethane 7010 elastomer (Deerfield Urethane Inc., South Deerfield,Mass.). The film demonstrates elastic behavior over a broad strainrange. No identifiable yield point is noted in this range and theminimal hysteresis on contraction is consistent with its spring-likebehavior. Similar results were obtained with a second elastomericpolyurethane sample.

Disadvantageously, polyurethane 7010 was found to sweat or crack whenexposed to common solvents used in biochemical assays, particularlysolvents such as ethanol and methanol, or chaeotropes such asguanidinium salts. The diaphragm material degrades within minutes,pneumatic integrity is impaired, and the diaphragm can cease to functionas a seal between the pneumatic and hydraulic subcavities. This behaviorrenders use of these polyurethanes problematic in certain molecularbiological assay cartridges.

FIG. 13 is a stress-strain curve obtained with a thin film of biaxiallyoriented polyethylene terephthalate (BoPET, sold under the trade nameMYLAR® by DuPont Teijin Films, Hopewell Va.). The material is verystiff, as indicated by the sharply elevated elastic modulus and theyield point above 100 MPa. The elastic limit of the material is exceededat about 5% deformation. However, the lack of deflection under operatingpressures likely to be useful in real-world devices also raises theissue as to whether a device having these materials can be reliablyself-priming. While BoPET is expected to be a tough material, it is ahigh modulus material and has a stiffness that is problematic in ayield-in-place process within the plastic body of a cartridge.

FIG. 14 is a stress strain curve for a PVDC/PET/PE co-laminate takenafter repeated cycles of strain and relaxation. As can be seen,following the first cycle (FIG. 11), subsequent cycles display little orno “memory” of the virgin film properties and shape. The slightestpressure results in a rapid ballooning of the film and essentially noelastic recovery is observed. This behavior reduces stroke response timeand resistance compared to elastomeric materials. In its stretched form,the film retains a suitable level of toughness for use in disposablecartridges of the invention.

FIG. 15 shows the effect of repeated cycles of strain and relaxation onBoPET. Once plastic deformation is achieved, the overstretched materialloses any resistance to serial application of pressure. On repeatedexercise of the film, essentially no force is necessary to distend thefilm (dashed line). However, the initial stretch step (solid line)requires a substantial application of force.

The size of features that can be formed in a material having a definedyield strength is dependent on the applied pressure or force such thatan increased degree of miniaturization that can be achieved by selectingmaterials with lower yield strengths. However, materials that have verylow yield strengths, such as those having a yield strength less than 2MPa, are likely to prove delicate and difficult to handle in amanufacturing environment and for that reason are not considered to begood candidates for making the locally stretched diaphragm webs of theinvention.

FIGS. 16A and 16B are cross-sectional views of a microvalve 2600 in thebody of a microfluidic device, showing an “ON” (or “open”) and an “OFF”(or “closed”) configuration of a diaphragm web 2601. The valve body isformed of multiple layers which include outside capping layer 2602 and acore formed by fusion of a molded pneumatic plate member 2603, a viaplate member 2604, and a molded hydraulic plate member 2605 such as bydiffusion bonding, or by an optional ACA glue layer 2612. Diaphragm 2601is sandwiched between plates 2603 and 2604. The valve cavity consists ofa pneumatic cavity 2606 and a hydraulic cavity 2607 separated by thediaphragm. Two fluidic channels (2608, 2609) are shown entering thehydraulic cavity 2607 through dual ports in a valve seat; the ports areseparated by a valve sill 2610. In the closed position (FIG. 16B), thevalve diaphragm seats on the valve sill and is pressurized at pneumaticport 2611 to resist flow of fluid from one channel to another. In theopen position (FIG. 16A), the diaphragm is retracted into the pneumaticcavity 2808 and fluid is free to flow across the valve sill from channel2608 to 2609. Movements of the diaphragm are actuated by a pneumaticsubcircuit ported into the pneumatic cavity at port 2611.

In other words, the valve includes a) a plastic body with internal valvecavity, the valve cavity being defined by a first enclosing lowersurface and a second enclosing upper surface, where the first surfacedefines a valve seat and the second surface sealingly apposes the firstsurface at a lip 2620 bounding the valve cavity; b) a diaphragm memberwith apron 2621 peripherally defined therearound, wherein the apron issealedly inserted into the body under the lip to separate the firstsurface from the second surface; c) a first fluidic channel entering thevalve cavity through the valve seat at a first port; d) a second fluidicchannel entering the valve cavity through the valve seat at a secondport; e) a valve sill 2810 defined on the first surface between thefirst port and the second port; and further wherein the diaphragm memberis capable of being reversibly deflected against and retracted from thevalve sill, thereby defining an “OPEN” position and an “OFF” positionfor allowing or not allowing flow of a fluid between the first channeland the second channel.

FIG. 17 is a cutaway perspective view of the valve structure of FIG. 16.In this instance the footprint of the valve has a roughly “peanut” shapewith an obvious waist narrowing in proximity to the valve sill 2610. Thediaphragm is shown as a partially distended, half peanut-shaped bulbwithin the valve cavity.

The peanut shape can be seen more clearly in FIG. 18A. The diaphragmmember 2601 is bilobate and includes an apron 2621 peripherally disposedaround the central bulbs. Positions of the inlet and outlet ports (whichare not part of the diaphragm) are indicated as dotted lines (2608 a,2609 a). FIG. 18B is an elevation/perspective view of a diaphragm member2601 with apron 2621 for a fluidic microvalve. The surface area of thestretched web approximates the interior wall surface area of thepneumatic subcavity 2606. The valve diaphragm is stretched into abulbous or “blister” shape (conforming generally to the internal cavityin which it is formed) after depressurization and, unlike valves formedwith an elastomeric diaphragm, may be advantageously supplied in the“OPEN” position in which there is essentially no resistance to fluidflow in the hydraulics of the device (i.e., from channel 2608 to 2609).Application of positive pressure through the pneumatic control line 2611collapses the diaphragm against the valve seat 2610 and turns the valve“OFF”.

FIG. 19 is an exploded view of the valve device 2600 of FIGS. 16-18. Bystretching a suitable diaphragm material, the footprint of the valvebody can be further miniaturized. Because of the pliancy ofLDPE/EVA/PVDC/EVA/LDPE and other stretch wrap films, for example, verysmall diaphragm features can be prestressed using the yield-in-placeprocess. Shown are stretched diaphragm 2601 with peripheral apron 2621,capping layer 2602, pneumatic layer 2603 with pneumatic cavity 2606, vialayer 2604, hydraulic layer 2605, with inlet and outlet channels (2608,2609) as marked.

The depth of the valve cavity 2606 in the z-dimension is exaggerated forpurposes of illustration. Valves of this type may be manufactured in the“OPEN” position, but can be reversibly closed at high speed (i.e., withreduced latency) by applying a pneumatic pressure of 2-12 psi throughthe pneumatic control line 2611. The valve diaphragm is stretched into athree-dimensional blister shape by application of pressure duringmanufacture, as in a yield-in-place process described with reference toFIG. 5, or by mechanical action as part of assembly or pre-assembly. Inthe yield-in-place process, pressure is applied through the fluidiccircuit or by applying negative pressure through the pneumatic circuitso as to yield and conform the polymeric film to the bulbous interiorcontour of the pneumatic subcavity 2606. Alternate mechanical processesfor pre-stretching the web will be described below.

By selection of a suitable film, and by adjustment of the conditions foryield-in-place stretching of the film during the manufacturing process,valves of this type having valve seats of less than about 0.5 mm inlength and 0.3 mm in width are readily obtained. Referring to theyield-in-place process, preferred films for millimeter-sized valvesinclude linear low density polyethylene (particularlymetallocene-catalyzed LLDPE), low density polyethylene blends andco-laminates generally, polyethylene vinyl acetate copolymers andlaminates, polyvinylidene chloride and PVDC co-polymer and laminates,and selected polyolefin composites and laminates, while not limitedthereto. With a suitable film, pneumatic valve features in thesub-millimeter scale are approachable. Generally, films having yieldstrengths of less than 15 or 20 MPa under manufacturing conditions arepreferable for making smaller valve features. A particularly preferredrange is 5 to 20 MPa; and for some applications 2 to 15 MPa. While yieldstrengths are cited for films under standard test conditions, it isunderstood that increased process temperature may also be used tooptimize conditions for manufacture of microvalves by this method.

As shown in FIG. 20, alternate valve structures 2800 may be formedhaving yielded diaphragm webs. The process of stretching the web toconform to an internal surface of the valve cavity may be used to makezero deadspace valves that are fluidically “OPEN” or are fluidically“OFF” when shipped. Initial stretching is generally achieved by applyinga positive pressure or force to the top of the diaphragm, forcing thediaphragm against the bottom surface of the bowl that forms the valveseat or land. The valve is shown in the “OFF” configuration in FIG. 20B.However, application of a pressure pulse to the fluid side (andoptionally zero or suction pressure on the pneumatic side), readilyallows the valve to open because there is essentially no elasticmodulus-related resistance as in prior art valves. Alternatively, asuction pressure can be applied to the pneumatic cavity, whichtransitions the valve back to the state or configuration shown in FIG.20A, but with the stretched diaphragm having a collapsed blister shapein which the surface area of the valve is greater than the upperinternal surface area of the valve chamber, thus resulting in a foldedirregular condition of the web which is readily reversible in theclosed, distended state. In our experience, these valves can withstandmany actuations and closures during a typical cartridge lifetime, whichis a few minutes to a few hours, and the deformation is not a hindranceto fluid flow in the “OPEN” state. Diaphragm materials in thesecartridges are generally yielded to form permanently overstretched filmstructures before release for sale, but may also be yielded in someembodiments during a preparatory step immediately before use in anassay.

In use, these valves may also be closed by applying a suction pressureto a downstream fluid column or by applying a positive pressure to thepneumatics, thus draining and/or expelling any residual fluid volumefrom the valve cavity. Control of gas (venting) or fluid flow throughthe valve may be modulated by varying the ratio of pressure on thehydraulic and pneumatic sides of the diaphragm or by applying pulsatilewaveforms of alternating positive and negative pressures.

The valves may be constructed by lamination or by fusion of molded bodyparts. Shown here are top capping layer 2802, diaphragm 2801, pneumaticbody layer 2803, hydraulic body layer 2804, and bottom capping layer2805. Also shown are valve seat 2807, pneumatic cavity 2808, hydrauliccavity 2809, first fluidic channel 2810, second fluidic channel 2811 andpneumatic actuation circuit 2812. The dark arrow indicates fluid flowwhen the valve is in the “OPEN” position (FIG. 20A). The double arrowindicates transition from the “OPEN” position to the “OFF” position,where fluid flow is blocked by the distended diaphragm on the valve seat2807 (FIG. 20B).

The hydraulic body part 2803 and pneumatic body part 2804 are depictedas molded parts and are joined at the dashed line by the diaphragm layer2801, which may be a layer of BoPET, SARANEX, polyvinylidene chloride,or other thin film that is stretchable by the processes described here.Optionally, elastic thin films may be used. However, a pre-stretchedfilm is advantageously flaccid and is actuated without the inherentresistance of an elastomer. Hydraulic pressure in a liquid entering thevalve from port 2810 is sufficient to cause fluid flow in a fully openstate.

FIG. 21 is a view of a yielded, bilobately stretched diaphragm or“blister” diaphragm of a microdevice 2800, and the web of the diaphragmis surrounded by an apron 2815. The apron is pinched between the bodylayers (2803, 2804) around the edges to separate the hydraulic subcavityand the pneumatic subcavity (2808, 2809) of the microvalve. This blistershape is flaccid, and the work required to collapse the blister isnegligible relative to the work required to overcome the inertia of thefluid under microfluidic conditions.

FIGS. 22A and 22B introduce the concept of valve latency. The time ittakes to turn on and off a valve is important in all fluidic processes,particularly for diagnostic assays where mixing, heating, and reactionkinetics are dependent on rapid valve actuation. In this example, a 10psi sustained pressure on the pneumatic side of the diaphragm isresponsible for keeping the valve in the CLOSED state at the START ofthe timeline where the timeline is represented by arrow 2820 runningfrom left to right. By releasing the positive pressure and applying anegative 5 psi suction pressure on the pneumatic side of the diaphragmat time 2821, the valve begins to transition to the OPEN state. However,this transition is not instantaneous and the delay is termed the“latency time”. In addition to the obvious concerns about stickiness ofthe diaphragm against the valve land, there are also material stiffnessand elasticity that can slow valve opening. Thus as achieved byconventional arts, valve latencies can be about 100 ms. However, and asshown in FIG. 22B, by pre-treating the diaphragm by a stretchingprocess, valve latency is substantially reduced. Because the material ispre-stretched, its resistance to transitioning from an open valve stateto a closed valve state is negligible, and can occur with minimalpressure, either in response to a negative pressure in the pneumaticactuation manifold, or by a positive fluid pressure in the hydraulicworks. Latency in the example shown, using a SARANEX® diaphragm wasmeasured at about 100 ms prior to stretching but after pre-stretching(POST) was reduced to less than 20 ms to full opening. This is asurprising result and would not have been an obvious action of knownelements behaving in predictable ways.

A negative pressure in the hydraulic works or a positive pressure in thepneumatic works is generally sufficient to close the valve depicted inFIG. 20, for example. Moreover, these valves may advantageously be usedin valve logic trees having a zero pressure state (typically a vent toatmosphere on the pneumatic side). In short, the valves may be operatedto open passively and close actively, if desired, an advance in the artover conventional valves. Surprisingly, this innovation eliminates theproblem of sticky valves that can occur in stored product usingelastomeric or rigid diaphragm materials.

Thus in another aspect, a pneumohydraulic valve is provided wherein thevalve is OPEN with no resistance to hydrostatically driven fluid flowwhen said pneumatic cavity is at atmospheric pressure (i.e., thepre-stretched diaphragm is flaccid) and OFF or closed when pressurizedby a pneumatic pressure greater than a hydrostatic pressure in thehydraulic chamber (for example as shown in FIG. 20, the pre-stretchedvalve conforming to the features of the valve seat 2807 to fluidlyseparate the inlet and outlet ports). More broadly, in this aspect, theinvention comprises a pneumohydraulic valve having a pneumatic cavityand a hydraulic cavity separated by a yielded diaphragm film, whereinthe valve is OPEN with no resistance to hydrostatically driven fluidflow when said pneumatic cavity is at atmospheric pressure and CLOSED oroff when pressurized by a pneumatic pressure greater than a hydrostaticpressure at either the inlet or the outlet of the hydraulic chamber. Itwould be understood by those skilled in the art, that increasinghydrostatic pressure on a liquid in the hydraulic cavity would readilydisplace a flaccid diaphragm film, and conversely, increasing thepneumatic pressure relative to the hydraulic pressure would inflate thediaphragm to its stretched shape and displace any fluid from thehydraulic cavity.

We now introduce the concept of a critical web dimension L_(c) astheory, where the “web” of a diaphragm 2501 is modeled as a circularpressurized bulge (as shown pictographically in FIGS. 1A and 2A) andchordal length L is defined in reference to FIG. 3. The critical webdimension L_(c) is the minimal web length for which a defined pressurewill result in a defined overstretch. The smaller the web dimension, themore pressure required to achieve the desired stretch. The relationshipapproximates a parabolic curve in which a pressure in excess of 30 psiis needed to achieve deformation of a 2 mm web of a thin film havingselected material properties. The derivation generally follows Freund LB and S Suresh, 2004. Film buckling, bulging and peeling. In, Thin filmmaterials—Stress Defect Formation and Surface Evolution. CambridgeUniversity Press, pp 312-386. A circular web of a thin film was analyzedas it deforms into a spherical cap by the action of an applied pressureP. Assuming the thin film deforms with a constant radius (i.e., aspherical deformation) and a constant equi-biaxial strain throughout themembrane (i.e., the radial and circumferential strains are equal andconstant at all points in the film), the stress in the membrane can thenbe related to strain as a function of Young's modulus, Poisson's ratio,and the film thickness. Neglecting bending stress within the material(i.e., assuming a thin film), the stress at the circumference of thefeature can be related to the pressure. Strain is then calculated asL′/L, the change in arc length from a flat film to a distended film.Finally, the critical diameter L_(c) for a given pressure can be derivedanalytically in cases where the deflection of the membrane is much lessthan the diameter of the feature.

In FIG. 23, the critical web dimension L_(c) of a film having thecharacteristics of SARANEX® film is calculated and plotted against thepressure at which a 6% yield is expected to occur. As can be seen, asthe film dimension L is reduced, higher pressure is required to stretchthe film. Plastic deformation of SARANEX® of more than 6.0% is predictedonly if the diaphragm web spans a dimension greater than L_(c) for agiven pressure. For example, a yield-in-place feature of about 3 mm isexpected by stretching a SARANEX® diaphragm under 30 psi of appliedpressure, which approaches the internal pressure at which damage to alaminated microfluidic cartridge is expected.

However, the above analysis has several shortcomings: the assumption ofa spherical deflection and equi-biaxial strain are not valid fornon-circular geometry, and strain will not be constant throughout theweb in a real valve. Also, the assumption that the deflection issignificantly less than the feature size does not hold, which will leadto an inaccurate analytical result. Another shortcoming is theinsufficiency of the material data for Young's modulus, which is ratedependent, and Poisson's ratio. The strain rate used in standardizedmaterials testing is likely at least an order of magnitude less thanthat of a pneumatically actuated valve in operation. Lastly, as the filmdistends and thins, the stress for a given pressure will be higher dueto a reduced cross sectional area. This could push the estimate of L_(c)to higher pressures or larger diameters. In short, the engineering ishighly unpredictable and complex, and the behavior of working valvescannot realistically be achieved without experiment. Surprisingly, inspite of theory, we found experimentally, using pressures less than 20psi, that valve diaphragms may be formed from 1 mil SARANEX® thin films(having a yield point in the range of 12-14 MPa) in cavities havingdimensions of about 2×3 mm and geometry essentially as shown in FIG. 18.This finding was unexpected, but welcome, because 20 psi (at elevatedtemperature) is within the working limit for pressurization of laminateddevice bodies without damage. Overstretch achieved was greater than 6%by length. Our theoretical analysis had suggested that microvalves ofthis size could not successfully be made at pressures less than 30 psi,raising the concern that “yield-in-place” or “form-in-place” stretchprocessing in a laminated device body would be impractical because oflimitations on internal pressure that could be tolerated by the device.

FIG. 24 shows the results of an experimental study of overstretchbehavior of a LDPE/EVA/PVDC/EVA/LDPE co-laminate diaphragm sealed over apneumatic chamber that is about 2 mm by 3 mm. An initial pressure wasapplied to remove any initial looseness. Center point deflection wasmeasured optically before and after application of a pressure sufficientto cause plastic deformation of the film. The change in plastic strainΔε is calculated as

${\Delta \; \varepsilon} = {\sqrt{\left( {\delta_{5}^{2} + R^{2}} \right)} - {\sqrt{\left. \left( {\delta_{0}^{2} + R^{2}} \right) \right)}/\sqrt{\left( {\delta_{0}^{2} + R^{2}} \right)}}}$

where load deflections are measured optically before (δ₀) and after (δ₅)pressure treatment of the film. Three results (as increase in centerlinestroke displacement) are shown following pressure treatment at 10, 15and 18 psi. An approximate doubling of the membrane stretch was obtainedafter pre-treatment at 18 psi, significantly greater than at 10 psi,demonstrating that treatment with about 15-20 psi is sufficient foryield-in-place diaphragm manufacture using LDPE/EVA/PVDC/EVA/LDPEco-laminate and the valve cavity has a height above the valve seat ofabout 100 micrometers.

This demonstrates that the inventive process achieves surprisingly smallmicrovalve features, features having dimensions that would not have beenpredictably achieved by application of known methods. For thin filmshaving a yield strength of less than 30 MPa, standardized processpressure and temperature conditions can be selected to achievestretch-in-place fluidic features having a desired size range. And as acorollary to that finding, using films having yield strengths less than15 MPa will result in yet smaller pump and valve features, animprovement that enables increased miniaturization. A synergy isachieved by using materials having lower yield points as valvediaphragms, and stronger materials for structural members forming themicrovalve cavity and associated channels.

Although PET is not considered suitable as a thin film for manufactureof form-in-place microvalves due to its high yield stress, PET is usefulfor the manufacture of device bodies, and thin films of PET can beincorporated by mechanically stretching the material where diaphragmwebs are to be formed. Thus the invention is not limited to lowerstrength diaphragm materials. Where mechanical stretch processing iscontemplated, a broader range of yield strengths may be considered. Forexample, while not limited thereto, materials such as BoPET, having ayield strength of about 100 MPa, may be incorporated by a mechanicalstretch process as described in the following figures.

FIGS. 25A through 25F are sequential views of steps in a first processof mechanically stretching a diaphragm film in place. The figuresdescribe a process of building a microfluidic device enclosing a valveand a pump cavity, where both diaphragms are stretched by a mechanicpress prior to final assembly. FIG. 25A shows a first molded body member2900 having internal cavities and channels; and in FIG. 25B a lowercapping layer 2901 is added to seal the channels. Viewed incross-section are a hydraulic subcavity of a valve 2902 (with valve sillseparating two fluidic channels) and a hydraulic subcavity of a pump2903 (with an inlet and an outlet). This forms the hydraulic works ofthe device, and defines a hydraulic valve subcavity and pump subcavitywith internal pumping as depicted schematically. In FIG. 25C, a thinfilm sheet of a suitable diaphragm film 2904 is overlaid on top of thehydraulic subassembly, covering the valve and pump subcavities. Then,the thin film material is welded or tacked to the molded part and excessmaterial is stripped away in a laser cut-weld, resulting in the valveand pump diaphragm web members (2912, 2913) depicted in FIG. 25D. Thewelding and cutting step is performed using a laser on an X-Y table, forexample. In a next step (FIGS. 25E to 25F), mechanical fingers (2910,2911) having suitable dimensions are used to force (arrows) thediaphragm webs into the hydraulic subcavities under process conditionssuitable to overstretch the film past its yield point and permanentlydeform the webs into the shape of a blister. Here the completedprestretched diaphragm web blisters (2915, 2916) corresponding to astretched valve diaphragm web and a pump diaphragm web respectively, areshown in a distended state conforming to the shape of the internalsurface area of the hydraulic cavities. A roller of suitable durometercould also be used to perform this process step. Upper layers of thedevice may then be glued or otherwise fused or affixed to the hydraulicsubassembly, sandwiching the diaphragm member in the completed devicebody. Final assembly is not shown, but would be understood by oneskilled in the art and in reference to FIG. 7 and FIG. 19, for example.

FIGS. 26A through 26F are sequential views of steps in a second processof mechanically stretching a diaphragm film in place. FIG. 26A shows afirst molded body member 2900 having internal cavities and channels; andin FIG. 26B a lower capping layer 2901 is added to seal the channels.Viewed in cross-section are a hydraulic subcavity of a valve 2902 (withvalve sill separating two fluidic channels) and a hydraulic subcavity ofa pump 2903 (with an inlet and an outlet). This forms the hydraulicworks of the device, and defines a hydraulic valve subcavity and pumpsubcavity with internal pumping as depicted schematically. In FIG. 26C,a thin film sheet of a suitable diaphragm film 2904 is overlaid on topof the hydraulic subassembly, covering the valve and pump subcavities.Then, the thin film material 2904 is welded or tacked to the molded partand excess material is stripped away in a laser cut-weld, resulting inthe valve and pump diaphragm members (2912, 2913) depicted in FIG. 26D.In this example, the cutting and fusion process is performed with anannular knife 2905 (or other suitable shape). Optionally the knife isheated or is sonically actuated so as to fuse the apron of the diaphragmmembers to the substrate. In a next step (FIGS. 26E to 26F), mechanicalfingers (2910, 2911) having suitable dimensions are used to force(arrows) the diaphragm webs into the hydraulic subcavities under processconditions suitable to overstretch the film past its yield point andpermanently deform the webs into the shape of a blister. Here thecompleted prestretched diaphragm web blisters (2915, 2916) correspondingto a valve diaphragm web and a pump diaphragm web respectively, areshown in a distended state conforming to the shape of the internalsurface area of the hydraulic cavities. A roller of suitable durometercould also be used to perform this process step. Upper layers of thedevice may then be glued or affixed to the hydraulic subassembly,sandwiching the diaphragm member in the completed device body.

The teaching of the invention is not limited to valves and pumps, butrelates to stretched diaphragms having a variety of functions inmicrofluidic devices. In the next example, we show a vent having amicroporous diaphragm, the vent occupying the terminus of a channel. Byselecting a hydrophobic microporous film, liquid may enter the hydrauliccavity while air is vented, allowing the cavity to fully fill withliquid.

Venting Elements Having Breathable Diaphragms

FIGS. 27A and 27B are representations of a close-ended micropump cavity3000 which finds use in a fluidic circuit having a branch thatterminates in a chamber 3004 with no outlet. Fluid enters the chambershown here through an inlet 3002 and fills the hydraulic subcavity 3004.In chambers of this type having diaphragms of the prior art, theresident gas cannot be displaced. However, by supplying a diaphragm web3001 of a breathable microporous film, gas is voided during the liquidfilling process through the diaphragm, through pneumatic subcavity 3005and out port 3006. By “breathable” is meant a film having permeabilityto gas but not to liquid. The diaphragm may then be pneumaticallyactuated to expel the fluid from the chamber, because the diaphragm,once wetted, no longer vents gas robustly but instead is transformedinto an efficient pumping member when actuated pneumatically. This novelfeature combines a microporous vent and a pump into an integral unitwith a single diaphragm. Port 3006 serves as a vent to outsideatmosphere during the fill cycle, but may be valved so as to be operatedby positive or negative pressure when operating diaphragm 3001 as apump. Inlet port 3002 also serves as the liquid outlet.

In application, a series of chambers in a fluidic device are fluidlyconnected to perform an assay, the end-terminal chamber (FIG. 27) of theseries having a breathable diaphragm 3001 that serves as a vent, so thatany air trapped in the dead end chamber may be removed during the filloperation. By pressurizing pneumatic cavity 3005, liquid is expelledback out the inlet 3002 and returned to the upstream fluidic circuit.

Micropumps of this kind can also be used for reagent additions where adried reagent is stored in the chamber for wetting at time of use, andfor thermocycling, for example, where a pair of pumps are slaved so thatone is actuated pneumatically, and the second is a close-ended fluidicbranch having a terminal chamber that is filled under pressure (whileventing gas through a microporous diaphragm) and then can be operated asa pneumatic pump to eject the liquid when acted on by a pneumaticoverpressure.

FIG. 28 depicts construction of a vented micropump element bylamination. The material may be pre-stretched by applying a pressuredifferential across its interface as described with reference to FIGS. 4and 5 or by using the mechanical stretch/assembly processes illustratedin FIGS. 25 and 26.

In another application, as shown in the device of FIG. 29A, breathablemicroporous films 3021 that have been stretched may be used to form aflow-through degassing 3020 chamber. This chamber includes an inlet 3022and an outlet 3023 for enabling flow of a liquid, but is topped by apneumatic subchamber separated from the hydraulics by a microporousmembrane 3021 (dashed line) through which gas can be withdrawn throughport 3024 under a pressure differential. Films formed in this way can beused for degassing small volumes of fluids. Breathable microporous filmscan also be used in chambers 3030 configured to add gas as shown in FIG.29B, such as for oxygenation of cells in flow cytometric applications orfor introduction of bubbles through thin film 3031 (dashed line) into afluid channel as where it is desirable to reform flow into a stream ofalternating fluid and bubble segments, as was achieved at a macroscopicscale in the AutoAnalyzer II, where bubbles were introduced at a “tee”into tubing carrying assay fluids at regular intervals to disrupt theunstirred boundary layer as is problematic in some microfluidicapplications. These films may be pre-stretched by the teachings of theinvention to reduce resistance to fluid flow and to increase surfacearea for gas exchange.

FIGS. 30A, B and C are electron micrographs of the fine structure of abreathable microporous polyurethane film, such as is useful in thedevices of FIGS. 28 and 29. A porous, fractured cellular structure isreadily visible with increasing magnification by scanning electronmicroscopy. Microporous polyurethanes include films sold as “PORELLE®”membranes (PIL Membranes Ltd, Kings Lynn, Norfolk UK). Thesepolyurethanes can preferably be hydrophobic, but hydrophilic films mayalso be useful. One example is Porelle 355.

Other microporous polymers are also known and function analogously.Microporous forms of polytetrafluoroethylene (PTFE) sold under the tradename MUPOR® (Porex, Fairburn Ga.) are readily yielded in place usinghydraulic pressure. A yield point of 2.2 MPa was determinedexperimentally for a 1 mil PFTE film under standard ASTM testconditions. The resulting diaphragms have good permeability to gas andcan be used as vents, and the hydrophobicity results in selectiveblockage of aqueous liquids if desired. In an unexpected solution to atechnical problem, microporous polyurethane films may thus be used toform diaphragm members in closed-end channels, where ingress of liquidinto a terminal chamber is possible only by directly venting theresident air through a permeable diaphragm. In some applications, thesediaphragms initially release air, but when wetted, permeability to airis substantially decreased, thus the diaphragm to a zero-air entrapment,self-priming pump for close-ended channels, where advantageously thepump becomes an active pneumatic micropump once all air in the line isvented and the film is wetted.

Application of dry pneumatic pressure or hydraulic pressure issufficient to cause films having a yield strength in the range of 2-30MPa to yield. The films will generally conform to the shape of aninternal cavity. Suitable form-in-place processes ensure that the fullvolume of the cavity is available for a subsequent pump stroke in thepresence of liquid, and is useful for example, when pump chambers areused in pairs, such as for two-zone thermocycling, particularly when oneof the pump chambers is a terminal chamber and is not otherwise vented.Alternatively, mechanical yielding processes of the invention may beused to form these yielded diaphragm members.

Circuit Combinations of the Inventive Fluidic Diaphragm Elements

FIG. 31 illustrates a representative fluidic circuit 3040 having acombination of diaphragm-operated circuit elements of the invention, asshown by drawing the channels and chambers without supporting substrate.This fluidic circuit consists of an inlet port 3041, a degassing chamber3042 connected to a sanitary vent 3043, two paired diaphragm pumps(3044,3045) for back-and-forth fluid flow, a detection chamber 3046 withoptical windows on the long axis and a terminal vent 3047. In operation,sample is added with aspiration, and is then directed through thecircuit by coordinated action of pneumatic actuators on diaphragmswithin each circuit element. In an assay, dried reagents (circles)prepositioned in the hydraulic subcavities result in chemical reactionsleading to a detection endpoint. Also needed for operation are twovalves (3048, 3049) that are used to fluidically isolate the paireddiaphragm pumps during reciprocating flow as in PCR amplification, whereone diaphragm pump is held at an annealing temperature and the otherdiaphragm pump at a denaturing temperature, as is known in the art.Advantageously, pre-stretched diaphragm members achieve improved pumpstroke volumes, improved consistency of stroke volume, reduce the workof pumping, and also improve the speed of the valves. Use of a stretchedmicroporous diaphragm for venting bubbles in the liquid is alsocontemplated and has been shown to offer advantages when fillingclose-ended channels. These circuit combinations having irreversiblystretch-deformed diaphragms are also conceived and claimed as part ofthe scope of this invention.

FIG. 32 illustrates a duplex microfluidic cartridge 3060 formed ofpneumatic and hydraulic circuits containing microvalves and micropumpsof the invention. While the detailed operation of these circuits isbeyond the scope of the description needed to appreciate the invention,one skilled in the art will understand that the inventive micropumps,microvalves, and microvents may be combined in larger assemblies of one,two or more microfluidic card devices and act cooperatively to functionin biological assays, including extractions and purifications, and alsoamplification and detection steps, and are capable of performingmultiple simultaneous functions with very small fluid volumes. In thisexample, the larger of the two devices 3061 is an extraction subcircuit,and the smaller device 3602 is a detection subcircuit. Also shown is agasket 3063 used in interfacing the microfluidic device pair through acommon manifold to an off-card pneumatic actuation circuit underinstrument control. To meet the challenge of higher throughput and assaycomplexity, increased miniaturization of these combinations is needed.The inventive diaphragms and sub combinations thereof result in improvedminiaturization and increased circuit density, as is a desirable advancein the art.

FIG. 33 tabulates yield point for several common films useful in themicrovalve diaphragms of the invention. Shown are yield points ofexemplary stretchable diaphragm film materials, including linear lowdensity polyethylenes, low density polyethylenes, ethylene vinyl acetatecopolymers, polyvinylidene chloride, and SARANEX®. High densitypolyethylenes have a marginally higher yield strength but are readilymodified to be suitable by a process of blending, grafting orco-lamination by skills known in the art. Suitable films for theyield-in-place process have yield strengths in the range of 2 to 30 MPa,but films having higher yield points may be used with the mechanicalprocesses described herein.

For example, also listed in the table for comparison is biaxiallyoriented polyethylene terephthalate, which has a narrow range ofelasticity (elastic modulus of 1.35 GPa) and a yield stress of about 100MPa. Films having a yield stress greater than 30 MPa are not generallypractical for yield-in-place processes at pressures and temperaturessuitable for manufacturing of micropumps and microvalves unless modifieddue to practical constraints on applied pressure and temperaturetolerated by the plastic cartridge body. Such films may be yielded usingmechanical presses or rollers as described earlier, or equivalentprocesses known in the art.

Polycarbonates have relatively high yield strengths (in the range of55-65 MPa) and elastic modulus (about 2.3 GPa). These materials areexpected to resist elongation and are not generally suitable foryield-in-place operations within a cartridge body, but may be blended orlaminated with more compliant materials, and may be stretchedmechanically under suitable conditions.

Polyimides are generally stiff materials with an elastic modulusexceeding 4 GPa and a yield strength of more than 70 MPa. Whileunstretched polyimides have been used as diaphragms in microfluidiccartridges, their inherent stiffness is not compatible with reliableself-priming features and implies higher operating pressure than aregenerally practicable unless blended or otherwise mechanically orpneumatically stretched prior to assembly to attain a suitable degree offlexibility.

Polyether ether ketone (PEEK) is generally not suitable. In addition toa yield stress of above 100 MPa, the Young's modulus is greater than 3.6GPa, indicating an extremely stiff material that will not readilystretch without substantial applied force.

PTFE has no memory and is not an elastomer. However, the yield strengthis relatively low (slightly more than 2 MPa). Surprisingly, microporousforms of PTFE sold under the trade name MUPOR® are readily yielded inplace using pneumatic pressure. These breathable films retain asignificant plasticity after yield point is exceeded, and can bestretched to conform to a chamber using pressures in a range suitablefor manufacturing of microassay cartridges. Microporous PTFE diaphragmsthat have been yielded in place may be operated as pumps or valves inthe devices of the invention when wetted.

A large range of polyolefinic and related plastics have been found to beuseful in forming stretch wraps and have yield strengths, toughness, andbonding characteristics suitable for use in the inventive microvalvesand micropumps. Of particular interest are acrylates, vinyl chlorides,biaxially oriented polypropylene, and esters, for example.Polyvinylchloride may be used in blends and co-laminates. Use ofpolyolefins as blends and co-laminates to form “stretch wrap” filmshaving the preferred yield strengths and bonding characteristics is wellknown in the art.

While the above is a description of the preferred embodiments of thepresent invention, it is possible to use various alternatives,modifications, combinations, and equivalents. Therefore, the scope ofthe present invention should be determined not with reference to theabove description but should, instead, be determined with reference tothe appended claims, along with their full scope of equivalents. Theappended claims are not to be interpreted as includingmeans-plus-function limitations, unless such a limitation is explicitlyrecited in a given claim using the phrase “means for.”

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent literature and publications referred to in this specificationand/or cited in accompanying submissions, including but not limited toU.S. Patent Application No. 61/745,340, are incorporated herein byreference, in their entirety. Aspects of the embodiments may bemodified, if necessary to employ concepts of the various patents,applications and publications to provide yet further embodiments. Theseand other changes may be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the specifics of the disclosure.

What is claimed is:
 1. A microfluidic cavity comprising a movable filmseparating two subcavities of the microfluidic cavity, said film havinglow elasticity, wherein the surface area of the film is larger than thecross-sectional area of the microcavity.
 2. The microfluidic cavity filmof claim 1, wherein the surface area of the film conforms in size to theinner surface area of one subcavity of said microcavity.
 3. Themicrofluidic cavity of any one of claims 1-2, wherein said film isselected from: a) a colaminate comprising a polyethyleneterephthalate/vinylidene chloride sandwich disposed between layers ofpolyethylene; b) a polyethylene, a polyvinylidene chloride, apolyolefin, an ethylene vinyl acetate, and co-laminates and compositesthereof; or, c) a high density polyethylene, polyethylene terephthalate,a polycarbonate, a polyimide, a polyether ether ketone, a polypropylene,a polyvinylchloride, a polyacrylate, a polyester, and co-laminates andcomposites thereof.
 4. The microfluidic cavity of any one of claims 1-3,wherein said film is a stretch wrap film, a co-laminate, or a compositethereof.
 5. The microfluidic cavity of any one of claims 1-3, whereinsaid film is a microporous polymer.
 6. The microfluidic cavity of anyone of claims 1-5, wherein said two subcavities of said microfluidiccavity define a valve, said valve comprising i) a first subcavityconfigured with a valve inlet, a valve outlet, and a valve seatinterposed between said valve inlet and said valve outlet, wherein saidfirst subcavity is configured to receive a fluid; and, ii) a secondsubcavity configured to be reversibly pressurized.
 7. The microfluidiccavity of claim 6, wherein said valve is fluidly joined to amicrofluidic circuit.
 8. The microfluidic cavity of any one of claims1-5, wherein said two subcavities of said microfluidic cavity define apump, said pump comprising i) a first subcavity configured to receive afluid; and, ii) a second subcavity configured to be reversiblypressurized.
 9. The microfluidic cavity of claim 8, wherein said pump isfluidly joined to a microfluidic circuit.
 10. A method of making a filmfor separating microfluidic cavities in a microfluidic cartridge, themethod comprising creating a low elasticity film by stretching a filmbeyond its yield point.
 11. The method of claim 10, wherein the film isstretched in place within the microfluidic cartridge.
 12. The method ofany one of claims 10-11, wherein the film is stretched in themicrofluidic cartridge during manufacture.
 13. The method of any one ofclaims 10-11, wherein the film is stretched in the microfluidiccartridge after manufacture.
 14. The method of claim 13, wherein thefilm is stretched in the microfluidic cartridge during initialoperation.
 15. The method of any one of claims 10-14, wherein the filmis stretched by the application of a sufficiently large pressuredifference to yield the film.
 16. The method of any one of claims 10-15,wherein the microfluidic cartridge is fully assembled.
 17. The method ofany one of claims 10-15, wherein the microfluidic cartridge is partiallyassembled.
 18. The method of claim 16, wherein the film is stretched bya pressure difference.
 19. The method of claim 17, wherein the film isstretched by a mechanical force.
 20. The method of claim 10, wherein thefilm is stretched by a die and then assembled into the microfluidicassembly.
 21. The method of claim 10, wherein the film is stretched andthen gathered into a shape complimentary to the microfluidic cavityusing an external die.
 22. The method of claim 10, wherein the film isstretched and then gathered into a shape complimentary to themicrofluidic cavity using the microfluidic cavity.
 23. A micropump, saidmicropump comprising a cavity having i) a first subcavity configured toreceive a fluid; ii) a second subcavity configured to be reversiblypressurized; iii) a diaphragm interposed between and separating saidfirst subcavity from said second subcavity; and, wherein said diaphragmis a polymeric thin film web having a yield point and is characterizedby a permanently overstretched deformation of said web.
 24. Themicropump of claim 23, wherein said thin film web is a low densitypolyethylene/ethylene vinyl acetate/polyvinylidene chloride/ethylenevinyl acetate/low density polyethylene co-laminate film.
 25. Themicropump of claim 23, wherein said thin film is composed ofpolyethylene, polyvinylidene chloride, poly-ethylene vinyl acetate,polyvinylchloride, polypropylene, polyolefin, or a composite or aco-laminate thereof.
 26. The micropump of claim 23, wherein said thinfilm web is a high density polyethylene, polyethylene terephthalate, apolycarbonate, a polyimide, a polyether ether ketone, a polypropylene, apolyvinylchloride, a polyacrylate, a polyester, or a co-laminate or acomposite thereof.
 27. The micropump of claim 23, wherein said thin filmweb is a stretch-wrap film.
 28. The micropump of claim 23, wherein saidthin film web is a chemically resistant polymer.
 29. The micropump ofany one of claims 23-28, wherein said micropump is self-priming.
 30. Themicropump of any one of claims 23-29, wherein said micropump ismanufactured by a process of locally overstretching said web under aforce applied to said thin film, said force being in excess of saidyield point.
 31. The micropump of any one of claims 23-30, wherein saidthin film web inelastically conforms in a first state to a firstinternal surface of said cavity when pressurized and in a second stateto a second internal surface of said cavity when depressurized.
 32. Themicropump of claim 31, wherein said micropump is configured to pump aliquid according to a pump stroke defined by the reversible motion ofsaid permanently overstretched deformation of said web between saidfirst state and said second state as driven by pressurization anddepressurization of said second subcavity.
 33. The micropump of claim32, wherein said first subcavity comprises a port configured forreceiving a liquid.
 34. The micropump of claim 32, wherein said firstsubcavity comprises a port for discharging a liquid.
 35. The micropumpof any one of claims 23-34, wherein the micropump is enclosed in adisposable cartridge body of a microassay device.
 36. The micropump ofclaim 30, wherein said force is a pneumatic force applied to saiddiaphragm after assembly of the disposable cartridge body.
 37. Themicropump of claim 30, wherein said web is first overstretched bylocally applying a force; and then said cavity is assembled by aligningsaid web with said cavity while apposingly mating said first subcavitywith said second subcavity.
 38. The micropump of claim 30, wherein saidweb is overstretched by a process of forcing said web into a subcavity;and then said cavity is assembled by apposingly mating said firstsubcavity with said second subcavity.
 39. The micropump of claim 30,wherein said force is a mechanical force applied under controlledprocess conditions.
 40. A microvalve, said microvalve comprising acavity having i) a first subcavity configured with a valve inlet, avalve outlet, and a valve seat interposed between said valve inlet andsaid valve outlet, wherein said first subcavity is configured to receivea fluid; ii) a second subcavity configured to be reversibly pressurized;iii) a diaphragm interposed between and separating said first subcavityfrom said second subcavity, wherein said diaphragm is enabled to bereversibly deflected against said valve seat according to whether saidsecond subcavity is pressurized or depressurized, thereby defining an“ON” position and an “OFF” position of said microvalve; and, furthercharacterized in that said diaphragm is a polymeric thin film web havinga yield point and is characterized by a permanently overstretcheddeformation of said web.
 41. The microvalve of claim 40, wherein saidthin film web is a low density polyethylene/ethylene vinylacetate/polyvinylidene chloride/ethylene vinyl acetate/low densitypolyethylene co-laminate film.
 42. The microvalve of claim 40, whereinsaid thin film is composed of polyethylene, polyvinylidene chloride,poly-ethylene vinyl acetate, polyvinylchloride, polypropylene,polyolefin, or a composite or a co-laminate thereof.
 43. The microvalveof claim 40, wherein said thin film web is a high density polyethylene,polyethylene terephthalate, a polycarbonate, a polyimide, a polyetherether ketone, a polypropylene, a polyvinylchloride, a polyacrylate, apolyester, or a co-laminate or a composite thereof.
 44. The microvalveof claim 40, wherein said thin film web is a stretch-wrap film.
 45. Themicrovalve of claim 40, wherein said thin film web is a chemicallyresistant polymer.
 46. The microvalve of any one of claims 40-45,wherein said microvalve is manufactured by a process of locallyoverstretching said web under a force applied to said thin film, saidforce being in excess of said yield point.
 47. The microvalve of claim46, wherein said force is a pressure applied under controlled processconditions.
 48. The microvalve of claim 46, wherein said force is amechanical force applied under controlled process conditions.
 49. Themicrovalve of any one of claims 40-48, wherein said thin film webinelastically conforms in a first state to a first internal surface ofsaid cavity when pressurized and in a second state to a second internalsurface of said cavity when depressurized.
 50. The microvalve of claim49, wherein said microvalve is configured to open and close saidmicrovalve by the reversible motion of said permanently overstretcheddeformation of said web between said first state and said second stateas driven by pressurization and depressurization of said secondsubcavity.
 51. The microvalve of any one of claims 40-50, wherein saidfirst subcavity comprises a port configured for receiving a liquid. 52.The microvalve of any one of claims 40-50, wherein said first subcavitycomprises a port for discharging a liquid.
 53. The microvalve of any oneof claims 40-52, wherein the microvalve is enclosed in a disposablecartridge body of a microassay device.
 54. The microvalve of claim 46,wherein said force is a pneumatic force applied to said diaphragm afterassembly of the disposable cartridge body.
 55. The microvalve of claim46, wherein said web is first overstretched by locally applying a force;and then said cavity is assembled by aligning said web with said cavitywhile apposingly mating said first subcavity with said second subcavity.56. The microvalve of claim 46, wherein said web is overstretched by aprocess of forcing said web into a subcavity; and then said cavity isassembled by apposingly mating said first subcavity with said secondsubcavity.
 57. A microassay device having a disposable cartridge bodyenclosing a micropump in a cavity therein, said micropump comprising i)a first subcavity configured to receive a liquid from a fluidic circuit;ii) a second subcavity configured to receive a pneumatic pressure; iii)a pneumohydraulic diaphragm interposed between and separating said firstsubcavity from said second subcavity; and, wherein said diaphragmcomprises a thin film web of a breathable microporous elastomer.
 58. Themicroassay device of claim 57, wherein said cavity is formed at aterminus of a fluidic circuit fluidly joined to said first subcavity.59. The microassay device of claim 57 or 58, wherein said microporouselastomer is a breathable hydrophobic polyurethane.
 60. The microassaydevice of claim 57 or 58, wherein said microporous elastomer is abreathable hydrophilic polyurethane.
 61. The microassay device of claim57 or 58, wherein said pneumatic pressure is a positive pressure or asuction pressure.
 62. A microassay device having a disposable cartridgebody enclosing a self-venting micropump in a cavity therein, saidmicropump comprising a) a hydraulic subcavity, wherein said hydraulicsubcavity defines a first surface of said cavity; b) a pneumaticsubcavity, wherein said pneumatic subcavity defines a second surface ofsaid cavity; c) a diaphragm disposed in said cavity and separating saidpneumatic subcavity from said hydraulic subcavity; d) a fluidic channelentering said hydraulic subcavity through said first surface, whereinsaid fluidic channel is configured as the sole fluid inlet and outlet ofsaid hydraulic subcavity; e) a pneumatic channel or vent entering saidpneumatic subcavity through said second surface; and, wherein saiddiaphragm comprises a thin film web of a breathable polymer.
 63. Thedevice of claim 62, wherein said breathable polymer is a microporouspolyurethane.
 64. The device of claim 62, wherein said breathablepolymer is a microporous polytetrafluoroether.
 65. The device of any oneof claims 62-64, wherein said pneumatic channel is a vent and saiddiaphragm is an elastomer operative as a passive pump operating byelastically storing energy when distended under pressure and elasticallyreleasing energy when relaxed.
 66. The device of any one of claims62-65, wherein said diaphragm comprises a thin film web of a breathablepolymer
 67. The device of any one of claims 62-66, wherein saidpneumatic channel is operatively connected to a pneumatic pulsegenerator enabled to apply a train of suction pressure pulses to saiddiaphragm.
 68. The device of any one of claims 62-66, wherein saidpneumatic channel is operatively connected to a pneumatic pulsegenerator enabled to apply alternating positive and suction pressurepulses to said diaphragm.
 69. A device for exchanging a gas in a liquid,said device comprising a) a body having a cavity therein, said cavitycomprising i) a hydraulic subcavity, wherein said hydraulic subcavitydefines a first surface of said cavity; ii) a pneumatic subcavity,wherein said pneumatic subcavity defines a second surface of saidcavity; iii) a thin film web disposed in said cavity and separating saidpneumatic subcavity from said hydraulic subcavity; b) a circuit enabledto flow the liquid from a first fluidic channel to a second fluidicchannel through said hydraulic subcavity, wherein said fluidic channelsare fluidly connected to said hydraulic subcavity through ports in saidfirst surface; c) a pneumatic channel entering said pneumatic subcavitythrough a port in said second surface, wherein said pneumatic channel isenabled to admit or evacuate the gas; and, wherein said thin film web isa breathable polymer.
 70. The device of claim 69, wherein saidbreathable polymer is a microporous elastomer.
 71. The device of claim69, wherein said breathable polymer is a microporous polyurethane. 72.The device of claim 69, wherein said breathable polymer is a microporouspolytetrafluoroether.
 73. The device of any one of claims 69-72, whereinsaid pneumatic channel is configured to enable evacuating the gas fromthe pneumatic subcavity at a reduced pressure.
 74. The device of claim73, wherein the device is configured for degassing the fluid flowingunder the thin film web.
 75. The device of any one of claims 69-72,wherein said pneumatic channel is configured to enable admitting the gasinto the pneumatic subcavity at a positive pressure.
 76. The device ofclaim 75, wherein said pneumatic channel is configured to inject abubble into the flowing fluid.